Sp110, a polypeptide component of the nuclear body

ABSTRACT

Cloning and characterization of a full length cDNA encoding Sp110 (speckled 110), a novel 110 kDa polypeptide, is disclosed. It is disclosed that Sp110 is a component of the nuclear body, is expressed in leukocytes, and is also expressed in other types of cells, including endothelial cells, smooth muscle cells, liver cells and heart cells, after contact with certain cytokines. The disclosure also includes the following: Sp140 recruits Sp110 to the nuclear body, Sp110 functions as an activator of gene transcription, and Sp110 serves as a nuclear hormone receptor co-activator. Sp110 DNAs, polypeptides, antibodies are disclosed. Also disclosed are Sp110-related screening methods and clinical diagnostic methods.

FEDERALLY SPONSORED RESEARCH

Work on the invention was supported in part by National Institutes ofHealth grants AR-01866 and DK-051179. Therefore, the government hascertain rights in the invention.

TECHNICAL FIELD

This invention relates to molecular biology, biochemistry, cell biology,medicine and medical diagnostics.

BACKGROUND

The nuclear body is a multiprotein complex located within the nuclei ofcells. The nuclear body is also known as nuclear domain 10, PMLoncogenic domain, and the Kr body. Immunohistochemical stainingtypically indicates 5-30 nuclear bodies within a nucleus. The nuclearbodies appear as discrete, punctate regions. The number of nuclearbodies in the cell, and the intensity of antibody staining of thesestructures, increase in response to heat shock and viral infection, aswell as following exposure to interferons and heavy metals (Ascoli etal., J. Cell Biol. 112:785-795, 1991). The nuclear body appears to beinvolved in the regulation of gene transcription. Nascent RNA polymeraseII transcripts have been found within the nuclear body (LaMorte et al.,Proc. Natl. Acad. Sci. USA 95:4991-4996, 1998), and the nuclear body isa preferred site for transcription of viral genes (Ishov et al., J.Cell. Biol. 138:5-16, 1997).

Promyelocytic leukemia (PML) protein is a component of the nuclear body.PML protein is involved in several cellular processes. For example, PMLprotein regulates cell growth (Wang, et al., Science 279:1547-1 551,1998) and may mediate apoptosis (Wang, et al., Nature Genetics20:266-272, 1998; Quignon et al., Nature Genetics 10:259-265, 1998). PMLprotein also recuits cAMP response element-binding protein (theCREB-binding protein or CBP) to the nuclear body and functions as apotent nuclear hormone receptor co-activator (Doucas et al., Proc. Natl.Acad. Sci. USA 96:2627-2632, 1999).

In addition to its involvement in gene transcription, the nuclear bodyis a target of autoantibodies in the sera of patients who have primarybiliary cirrhosis (PBC), an autoimmune disease (Hodges et al., Am. J.Hum. Genet. 63:297-304, 1998; Melnick et al., Blood 93: 3167-3215, 1999;Sternsdorf et al., Immunobiology 198:307-331, 1997). PBC patients carryautoantibodies directed against Sp100 (Speckled 100 kDa), a polypeptidecomponent of the nuclear body (Szostecki et al., J. Immunol.145:4338-4347, 1990). Two splice variants of Sp100, designated Sp100band Sp100-HMG, have also been found (Dent et al., Blood 88:1423-1436,1996; Seeler et al. Proc. Natl. Acad. Sci. USA. 95:7316-7321, 1998;Lehming et al., Proc. Natl. Acad. Sci. USA 95:7322-7326, 1998). Theseproteins interact with members of the heterochromatin protein 1 (HP1)family of non-histone chromosomal proteins. When bound to a promoter,the Sp100 proteins and HP1 behave as transcriptional repressors intransfected cells. These observations suggest that the nuclear body ingeneral, and the Sp100 proteins in particular, may maintain chromatinarchitecture and regulate gene transcription (Seeler, et al. Proc. Natl.Acad. Sci. USA 95:7316-7321, 1998 and Lehming et al., Proc. Natl. Acad.Sci. USA 95:7322-7326).

Sera from PBC patients have also been used to identify aleukocyte-specific component of the nuclear body designated Sp140 (Blochet al., J. Biol. Chem. 46:29198-29204, 1996). The N-terminal portion ofSp140 exhibits sequence homology with the N-terminal segments of theSp100 proteins. The middle region of Sp140 contains a “SAND” domain(Gibson et al., Trends Biochem. Sci. 23:242-244, 1998), and theC-terminal portion of Sp140 contains a plant homeobox domain and abromodomain.

SUMMARY

A full length cDNA encoding Sp110 (Speckled 110), a novel 110 kDapolypeptide, has been discovered and characterized. It has beendiscovered that Sp110 is a component of the nuclear body, is expressedin leukocytes, and is also expressed in other types of cells, includingendothelial cells, smooth muscle cells, liver cells and heart cells,after contact with cytokines, including tumor necrosis factor,interleukin 1, and interferons. Other discoveries include the following:Sp140 recruits Sp110 to the nuclear body, Sp110 functions as anactivator of gene transcription, and Sp110 serves as a nuclear hormonereceptor co-activator.

Based on these and other discoveries, the invention features an isolatedDNA containing a nucleotide sequence whose complement hybridizes understringent hybridization conditions to a DNA molecule whose nucleotidesequence consists of nucleotides 405 to 797 of the Sp110 cDNA (SEQ IDNO:1). In some embodiments, the isolated DNA also includes at least oneof the following: a nucleotide sequence encoding a domain having atleast 80% sequence identity with amino acids 6-109 (Sp100-like domain)of the Sp110 polypeptide (SEQ ID NO:2); a domain having at least 80%sequence identity with amino acids 454-532 of SEQ ID NO:2 (SAND domain);a domain having at least 80% sequence identity with amino acids 537-577of SEQ ID NO:2 (plant homeobox domain); and a domain having at least 80%sequence identity with amino acids 606-674 of SEQ ID NO:2 (bromodomain).

In some embodiments, the DNA hybridizes as described above and includesa nucleotide sequence encoding at least one of the following: aminoacids 6-109 of SEQ ID NO:2 (Sp100-like domain) or amino acids 6-109 ofSEQ ID NO:2 with one or more, e.g., 5, 10, 15 or 20 conservative aminoacid substitutions therein; amino acids 454-532 of SEQ ID NO:2 (SANDdomain) or amino acids 454-532 of SEQ ID NO:2 with one or more, e.g., 5,10, 15 or 20, conservative amino acid substitutions therein; amino acids537-577 of SEQ ID NO:2 (plant homeobox domain) or amino acids 537-577 ofSEQ ID NO:2 with one or more, e.g., 5, 10, 15 or 20, conservative aminoacid substitutions therein; and amino acids 606-674 of SEQ ID NO:2(bromodomain) or amino acids 606-674 of SEQ ID NO:2 with one or more, 5,10, 15 or 20, conservative amino acid substitutions therein.

In some embodiments, the isolated DNA hybridizes as described above, andalso includes a nucleotide sequence encoding all of the following: aminoacids 6-109 of SEQ ID NO:2 (Sp100-like domain) or amino acids 6-109 ofSEQ ID NO:2 with one or more conservative amino acid substitutionstherein; amino acids 454-532 of SEQ ID NO:2 (SAND domain) or amino acids454-532 of SEQ ID NO:2 with one or more conservative amino acidsubstitutions therein; amino acids 537-577 of SEQ ID NO:2 (planthomeobox domain) or amino acids 537-577 of SEQ ID NO:2 with one or moreconservative amino acid substitutions therein; and amino acids 606-674of SEQ ID NO:2 (bromodomain) or amino acids 606-674 of SEQ ID NO:2 withone or more conservative amino acid substitutions therein.

In some embodiments, the isolated DNA contains a nucleotide sequencethat encodes a polypeptide whose amino acid sequence is the sequence setforth as SEQ ID NO:2 or the sequence set forth as SEQ ID NO:2, with oneor more, e.g., 5, 10, 15 or 20 conservative amino acid substitutionstherein.

In some embodiments, the isolated DNA hybridizes as described above andalso includes a nucleotide sequence encoding an Sp110 inhibitorpolypeptide containing: amino acids 6-109 of SEQ ID NO:2 (Sp100-likedomain) or amino acids 6-109 of SEQ ID NO:2 with one or moreconservative amino acid substitutions therein; amino acids 537-577 ofSEQ ID NO:2 (plant homeobox domain) or amino acids 537-577 of SEQ IDNO:2 with one or more conservative amino acid substitutions therein; andamino acids 606-674 of SEQ ID NO:2 (bromodomain) or amino acids 606-674of SEQ ID NO:2 with one or more conservative amino acid substitutionstherein; wherein the polypeptide does not contain a SAND domain.

In some embodiments, the isolated DNA contains a nucleotide sequence(Sp110 splice variant) that encodes the amino acid sequence set forth asSEQ ID NO:5, or the sequence set forth as SEQ ID NO:5 with one or more,e.g., 5, 10, 15 or 20, conservative amino acid substitutions therein.

The invention also features a vector containing any of the DNAsdescribed above, and a host cell containing the vector. In the vector,the DNA can be operably linked to one or more expression controlsequences.

The invention also features a substantially pure polypeptide encoded byany of the DNAs described above. In addition, the invention includes asubstantially pure polypeptide (an inhibitor of Sp110 activity)containing an Sp110 Sp100-like domain, an Sp110 SAND domain, an Sp110plant homeobox domain, and an Sp110 bromodomain, wherein the sequence ofamino acids 110 to 453 of SEQ ID NO:2 is not present. In someembodiments, this polypeptide includes a membrane transport moiety,i.e., a moiety that allows the polypeptide to enter a cell. Exemplarymembrane transport moieties are an internalization peptide sequencederived from Antennapedia and an HIV tat peptide.

The invention also features antibodies that bind specifically to theSp110 polypeptide. The antibodies can be labeled.

The invention also features a screening method for identifying acompound that inhibits Sp110 dimerization. The method, which can be invitro, includes: providing an Sp110 polypeptide sample solution; addingto the sample solution a candidate compound; and detecting a decrease inSp110 dimerization in the presence of the candidate compound, ascompared to Sp110 dimerization in the absence of the candidate compound

The invention also features a screening method for identifying acompound that enhances or promotes Sp110 dimerization. The method, whichcan be in vitro, includes: providing an Sp110 polypeptide samplesolution; adding to the sample solution a candidate compound; anddetecting an increase in Sp110 dimerization in the presence of thecandidate compound, as compared to Sp110 dimerization in the absence ofthe candidate compound.

The invention also features a screening method for identifying apolypeptide that dimerizes with Sp110 to form an inactive heterodimer.The method includes: providing an Sp110 polypeptide sample solution;adding to the sample solution a candidate polypeptide, thereby forming atest mixture; providing a gene expression system comprising a reportergene operably linked to an Sp110-responsive expression control sequence;contacting the test mixture with the gene expression system; anddetecting a decrease in reporter gene expression level in the presenceof the test mixture, as compared to gene expression level in thepresence of the Sp110 polypeptide sample solution. The gene expressionsystem can be in a living cell, e.g., a transformed host cell.Alternatively, the gene expression system can be in vitro, i.e.,acellular.

The invention also features a screening method for identifying apolypeptide that dimerizes with Sp110 to form a constitutively active orhyperactive heterodimer. The method includes: providing an Sp110polypeptide sample solution; adding to the sample solution a candidatepolypeptide, thereby forming a test mixture; providing a gene expressionsystem comprising a reporter gene operably linked to an Sp110-responsiveexpression control sequence; contacting the test mixture with the geneexpression system (which can be cellular or acellular); and detecting aconstitutive activity or an increase in reporter gene expression levelin the presence of the test mixture, as compared to gene expressionlevel in the presence of the Sp110 polypeptide sample solution.

The invention also features a screening method for identifying acompound or polypeptide that inhibits Sp110 binding to a nuclear hormonereceptor. The method, which can be in vitro, includes: providing anSp110 polypeptide sample solution; adding to the sample solution acandidate compound; adding to the sample solution a nuclear hormonereceptor; and detecting a decrease in Sp110 binding to the nuclearhormone receptor in the presence of the candidate compound, as comparedto Sp110 binding to the nuclear hormone receptor in the absence of thecandidate compound.

The invention also features a screening method for identifying acompound or polypeptide that enhances Sp110 binding to a nuclear hormonereceptor. The method, which can be in vitro, includes: providing anSp110 polypeptide sample solution; adding to the sample solution acandidate compound; adding to the sample solution a nuclear hormonereceptor, and detecting an increase in Sp110 binding to the nuclearhormone receptor in the presence of the candidate compound, as comparedto Sp110 binding to the nuclear hormone receptor in the absence of thecandidate compound.

The invention also features a screening method for identifying acompound or polypeptide that inhibits the binding of an Sp110 dimer toan Sp110-binding nucleotide sequence. The method, which can be in vitro,includes: providing an Sp110 polypeptide sample solution; adding to thesample solution a candidate compound or polypeptide; adding to thesample solution an Sp110-binding nucleotide sequence; and detecting adecrease in Sp110 binding to the Sp110-binding nucleotide sequence inthe presence of the candidate compound, as compared to Sp110 binding tothe Sp110-binding nucleotide sequence in the absence of the candidatecompound.

The invention also features a screening method for identifying acompound or polypeptide that enhances or promotes the binding of anSp110 dimer to an Sp110-binding nucleotide sequence. The method, whichcan be in vitro, includes: providing an Sp110 polypeptide samplesolution; adding to the sample solution a candidate compound orpolypeptide; adding to the sample solution an Sp110-binding nucleotidesequence; and detecting an increase in Sp110 binding to theSp110-binding nucleotide sequence in the presence of the candidatecompound, as compared to Sp110 binding to the Sp110-binding nucleotidesequence in the absence of the candidate compound.

The invention also features a method for diagnosing primary biliarycirrhosis (PBC) in a human patient. The method includes: providing asubstantially pure Sp110 polypeptide; providing a serum sample from thepatient; contacting Sp110 polypeptide with the serum sample; anddetecting specific binding of an antibody in the serum with the Sp110polypeptide as an indication of PBC.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. In case of conflict, thepresent application, including definitions, will control. Allpublications, patents and other references mentioned herein areincorporated by reference.

Although methods and materials similar or equivalent to those describedherein can be used in the practice or testing of the present invention,the preferred methods and materials are described below. The materials,methods and examples are illustrative only and not intended to belimiting. Other features and advantages of the invention will beapparent from the detailed description and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is the nucleotide sequence of a full-length, human Sp110 cDNA(SEQ ID NO:1)

FIG. 2 is a comparison of the deduced amino acid sequences of Sp110 (SEQID NO:2) and Sp140 (SEQ ID NO:3). Shaded portions of the sequencesindicate the Sp100-like domain, the SAND domain, the plant homeoboxdomain (PHD), and the bromodomain. Conserved cysteine/histidine residueswithin the PHD are marked with asterisks. A dashed box encloses apredicted nuclear localization sequence, and a solid box encloses theLXXLL-type nuclear hormone receptor interaction domain. The sequence ofthe interferon-inducible protein nuclear phosphoprotein 72 begins atamino acid-241 (met) and ends at amino acid 605 (leu), which areindicated by arrows.

FIG. 3 is the nucleotide sequence of the full-length, human Sp110 cDNA(SEQ ID NO:1) and the deduced amino acid sequence (reading frame c) ofthe Sp110 polypeptide (SEQ ID NO:2).

FIG. 4. is the nucleotide sequence of a human Sp110 splice variant cDNA(Sp110b) (SEQ ID NO:4) and the deduced amino acid sequence (readingframe c) of the Sp110b polypeptide (SEQ ID NO:5).

FIG. 5 is a schematic diagram comparing the domain structures of humanSp110, Sp140 and Sp100b. Also shown is the percent identity of the Sp110domains, as compared to the corresponding domains in Sp140 and Sp100b.

DETAILED DESCRIPTION

The full-length, naturally-occurring, human Sp110 polypeptide includesan Sp100-like domain (amino acids 6-109 of SEQ ID NO:2), a SAND domain(amino acids 454-532 of SEQ ID NO:2), a plant homeobox domain (PHD)(amino acids 537-577 of SEQ ID NO:2), and a bromodomain (amino acids606-674 of SEQ ID NO:2). The full-length Sp110 functions as an activatorof gene transcription. Sp110 also functions as a nuclear hormonereceptor co-activator. Some embodiments of the invention include all ofthe domains in a single Sp110 polypeptide. In other embodiments,however, one or more of the domains is modified or absent

The Sp100-like domain in the N-terminal portion of Sp110 has a potentialhelical motif, which can mediate homodimerization. (Seeler et al., Proc.Natl. Acad. Sci. USA 95:7316-7321, 1998). It is predicted that theSp100-like domain in Sp110 functions in protein binding interactions,e.g., dimerization, with Sp140 or Sp100 to form a heterodimer, ordimerization with a second Sp110 polypeptide to form a homodimer.Therefore, a polypeptide that contains an Sp110 Sp100-like domain (orderivative thereof), but does not contain an activating domain, can beused in a cell to form inactive dimers comprising Sp140, Sp100 or Sp110.Such formation of inactive dimers reduces the availability of endogenousSp140, Sp100 or Sp110 monomers for formation of active(transcription-activating or transcription-inhibiting) dimers, therebyreducing Sp140, Sp100, or Sp110 activity in the cell.

The Sp110 polypeptide is predicted to contain an activation domainlocated in the region between the Sp100-like domain and the SAND domain,i.e., in the region between amino acids 109 and 454 of SEQ ID NO:2. Insome embodiments of the invention, a polypeptide containing this regionor a derivative thereof is used to enhance or promote Sp110 activity.

Sp110 contains a SAND domain, variations of which are found in Sp100,Sp140, AIRE-1 (Nagamine et al., Nature Genetics 17:393-397, 1997),nuclear phosphoprotein 72, and DEAF-1 (Gross et al., EMBO J.15:1961-1970, 1996). It is predicted that the Sp110 SAND domain is aDNA-binding domain (Gibson et al., Trends Biochem Sci. 23:242-244,1998). In some embodiments of the invention, a polypeptide that containsa SAND domain (or derivative thereof), but lacks an activating domain,is employed as an inhibitor of Sp110 activity in a cell e.g., a cellcultured in vitro. An example is an Sp110 polypeptide lacking the regionextending from approximately amino acid 110 to amino acid 453 in SEQ IDNO:2. Such a polypeptide inhibits Sp110 activity because the SAND domainoccupies some or all of the Sp110 binding sites, thereby blocking accessof active Sp110 molecules to the binding sites. In some embodiments, amembrane transport moiety, e.g., the internalization peptide sequencederived from Antennapedia (Bonfanti et al., Cancer Res. 57:1442-1446) oran HIV tat peptide (U.S. Pat. No. 5,652,122), is conjugated to the SANDdomain to facilitate entry of the SAND domain into living cells. Inother embodiments, the carrier moiety is a polypeptide fused to the SANDdomain or polypeptide containing the SAND domain, e.g., at the aminoterminus of the SAND domain-containing polypeptide.

The full-length Sp110 polypeptide contains a plant homeobox domain(PHD). This is a cysteine-rich region that spans 50-80 amino acidresidues and contains the motif Cys₄-His-Cys₃ (Aasland et al., TrendsBiochem. Sci. 20:56-59, 1995). This motif is found in many proteins thatare involved in chromatin-mediated control of gene transcription. It ispredicted that the Sp110 PHD functions in protein-protein or protein-DNAinteractions.

The full-length Sp110 polypeptide contains a bromodomain. It ispredicted that the Sp110 bromodomain functions catalytically inacetylation of histones. The bromodomain is an a helical motif found inmany proteins involved in the regulation of gene transcription(Jeanmougin et al., Trends Biochem. Sci. 22:151-153, 1997). In general,the bromodomain is found in transcription factors that have catalyticdomains. For example, SW12/SNF2 has a DNA-dependent ATPase domain(Laurent et al., Genes Dev. 7:583-591, 1993), TAF_(II)250 (Dickstein etal., Cell 84:781-790, 1996) and TIF1α (Fraser et al., J. Biol. Chem.273:16199-16204, 1998) have kinase domains, and GCN5 has a histoneacetyl-transferase (HAT) domain (Brownwell et al., Cell 84:843-851,1996). The original description of the bromodomain reported a conservedmotif spanning approximately 60 amino acid residues and containing two ahelices (Haynes et al., Nucl. Acids Res. 20:2603, 1992). Subsequently ithas been suggested that the bromodomain spans 110 amino acid residuesand contains two additional α helices (Le Douarin et al., EMBO J.15:6701-6715, 1996). The four predicted a helices were designated Z, A,B, and C. The Sp110 bromodomain of SEQ ID NO:2 includes the A, B, and Chelices but lacks the Z helix. Some Sp110 splice variants may have a Zhelix.

Sp110 polypeptides, and fragments and derivatives thereof, can beobtained by any suitable method. For example, Sp110 polypeptides can beproduced using conventional recombinant DNA technology, as described inthe Examples below. Guidance and information concerning methods andmaterials for production of polypeptides using recombinant DNAtechnology can be found in numerous treatises and reference manuals.See, e.g., Sambrook et al, 1989, Molecular Cloning—A Laboratory Manual,2^(nd) Ed., Cold Spring Harbor Press; Ausubel et al. (eds.), 1994,Current Protocols in Molecular Biology, John Wiley & Sons, Inc.; Inniset al. (eds.), 1990 PCR Protocols, Academic Press.

Alternatively, Sp110 polypeptides or fragments thereof can be obtaineddirectly by chemical synthesis, e.g., using a commercial peptidesynthesizer according to vendor's instructions. Methods and materialsfor chemical synthesis of polypeptides are well known in the art. See,e.g., Merrifield, 1963, “Solid Phase Synthesis,” J. Am. Chem. Soc.83:2149-2154.

Percent identity between amino acid sequences referred to herein isdetermined using the BLAST 2.0 program, which is available to the publicat http://www.ncbi.nlm.nih.gov/BLAST. Sequence comparison is performedusing an ungapped alignment and using the default parameters (Blossom 62matrix, gap existence cost of 11, per residue gap cost of 1, and alambda ratio of 0.85). The mathematical algorithm used in BLAST programsis described in Altschul et al., 1997, Nucleic Acids Research25:3389-3402.

As used herein “isolated DNA” means DNA that has been separated from DNAthat flanks the DNA in the genome of the organism in the which the DNAnaturally occurs. The term therefore includes a recombinant DNAincorporated into a vector, e.g., a cloning vector or an expressionvector. The term also includes a molecule such as a cDNA, a genomicfragment, a fragment produced by PCR, or a restriction fragment. Theterm also includes a recombinant nucleotide sequence that is part of ahybrid gene construct, i.e., a gene construct encoding a fusion protein.

As used herein, “high stringency” means the following: hybridization at42° C. in the presence of 50% formamide; a first wash at 65° C. with2×SSC containing 1% SDS; followed by a second wash at 65° C. with0.1×SSC.

As used herein, “substantially pure polypeptide” means a polypeptideseparated from components that naturally accompany it. For example, apolypeptide is substantially pure when it is at least 80%, by weight,free from the proteins and other organic molecules with which it isnaturally associated. Purity can be measured by any suitable method,e.g., column chromatography, polyacrylamide gel electrophoresis, or HPLCanalysis. A chemically synthesized polypeptide or a recombinantpolypeptide produced in a cell type other than the cell type in which itnaturally occurs is, by definition, substantially free from componentsthat naturally accompany it.

As used herein, “conservative amino acid substitution” means asubstitution within an amino acid family. Families of amino acidresidues are recognized in the art and are based on physical andchemical properties of the amino acid side chains. Families include thefollowing: amino acids with basic side chains (e.g. lysine, arginine,and histidine); amino acids with acidic side chains (e.g., aspartic acidand glutamic acid); amino acids with uncharged polar side chains (e.g.glycine, asparagine, glutamine, serine, threonine, tyrosine, andcysteine); amino acids with nonpolar side chains (e.g. alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine, andtryptophan); amino acids with branched side chains (e.g., threonine,valine, and isoleucine); and amino acids with aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, and histidine). An aminoacid can belong to more than one family.

A full-length Sp110 polypeptide, Sp110 polypeptide fragments containingindividual Sp110 domains, or other antigenic Sp110 fragments, can beused to produce Sp110-specific antibodies. An example of an Sp110fragment that can be used to elicit Sp110-specific antibodies is apolypeptide consisting of amino acids 219-235 of SEQ ID NO:2. TheSp110-specific antibodies can be readily obtained, without undueexperimentation, through application of conventional techniques.

In view of its production by cells involved in host defense, i.e.,leukocytes, and its induction by interferon (IFN) and cytokines, Sp110appears to play a role in inhibiting viral replication and facilitatingdifferentiation of cells, e.g., myeloid cells, and activation of cellsinvolved in host defense. For example, an Sp110 polypeptide can be usedtherapeutically to treat myeloid malignancies by virtue of its abilityto promote myeloid cell differentiation. In addition, the nuclear body,of which Sp110 is a structural component, is disrupted in various humandisorders, including acute promyelocytic leukemia and viral infections.Therefore, in some embodiments of the invention, an Sp110 polypeptide(or derivative thereof) is introduced into a cell in vitro or in amammal to enhance cellular defense mechanisms. In other embodiments,Sp110 is administered therapeutically to treat inflammation or toachieve alteration in lipid profiles.

A preformed Sp110 polypeptide can be introduced into a cell usingconventional techniques for transporting proteins into intact cells,e.g., by using the polypeptide to the internalization peptide sequencederived from Antennapedia (Bonfanti et al., Cancer Res. 57:1442-1446) orto an HIV tat peptide (U.S. Pat. No. 5,652,122). Alternatively, theSp110 polypeptide can be expressed in the cell following introduction ofan Sp110-encoding DNA, e.g., in a conventional expression vector,according to the invention. In some embodiments, Sp140 is concurrentlyintroduced into the cell, or co-expressed in the cell, so that the Sp140can recruit the Sp110 into nuclear bodies.

The biological activities of Sp110 include co-activation of nuclearhormone receptors, e.g., retinoic acid receptors (RARs), RXRs, LXR, FXR,peroxisome proliferator-activated receptors (PPARs), including PPARα andPPARγ, glucocorticoid receptors, estrogen receptors, progesteronereceptors, androgen receptors, and orphan nuclear hormone receptors.Nuclear hormone receptors mediate signal transduction in variouscellular responses. Sp110 appears to enhance expression of nuclearhormone-responsive genes by binding to a nucleotide sequence adjacent tothe nuclear hormone response element and directly or indirectlyenhancing gene expression. Activities of RARs and RXRs are important incellular differentiation. Activities of PPARα and PPARγ are important infatty acid metabolism and inflammation. Activities of FXR and LXR areimportant for cholesterol metabolism.

Where increased co-activation of an Sp110-responsive nuclear hormonereceptor is needed, e.g., to enhance PPARα-mediated inhibition of theinflammatory response in smooth muscle cells or endothelial cells, anSp110 polypeptide can be supplied in a therapeutic method. Similarly, incardiac myocytes, Sp110 may augment PPARα's effect on lipid metabolism,potentially attenuating cardiac hypertrophy. Moreover, in adipocytesSp110 may alter PPARγ regulation of lipid storage, potentially treatingobesity.

Sometimes a nuclear hormone receptor-mediated response needs to belimited or reduced therapeutically. For example, an Sp110 derivative canbe used to inhibit FXR receptors, thereby enhancing conversion ofcholesterol to bile acids. In another example, Sp110 is used to blockestrogen receptors in treatment of estrogen responsive tumors. Theinvention includes inhibitors of Sp110 activity, screening methods foridentifying inhibitors of Sp110 activity, and methods of inhibitingSp110 activity in cells in vitro or in a mammal.

Inhibition of Sp110 activity can be accomplished through approachesincluding the following: a polypeptide that dimerizes with endogenousSp110 polypeptides to form an inactive dimer; a small molecule (MW=1000Da or less) that interferes with Sp10 dimerization; a polypeptide thatoccupies Sp110 binding sites (nucleotide sequences) in DNA withoutcausing transcriptional activation; and a small molecule that interfereswith Sp110-nuclear hormone receptor interactions.

An example of a polypeptide predicted to dimerize with endogenous Sp110polypeptides to form an inactive dimer is a polypeptide that includesamino acids 1-453 of the Sp110 sequence fused to amino acids 533-689 ofthe Sp110 sequence (amino acids 1-453 of SEQ ID NO:2 fused to aminoacids 533-689 of SEQ ID NO:2). Such a polypeptide includes the entireSp110 amino acid sequence except the Sp110 SAND domain, which ispredicted to be required for recognizing and binding to Sp110 bindingsites in DNA, but not required for Sp110 dimerization.

An example of a polypeptide predicted to recognize and occupy Sp110binding sites (nucleotide sequences) in DNA without causingtranscriptional activation is an Sp110 SAND domain or fragment thereof.

Polypeptides and other molecules, e.g., small molecules, that inhibit orpromote Sp110 activity can be identified by screening methods providedby the invention. The type of screening method employed will depend onthe type of inhibition mechanism chosen. One general approach is basedon Sp110 dimerization, which is predicted to be necessary for Sp110biological activity. One variation on this approach is to provide amolecule that interferes with Sp110 dimerization. Another variation onthis approach is to provide a polypeptide that dimerizes with endogenousSp110 polypeptides to form an inactive dimer. A second general approachis to provide a molecule that binds to (blocks) a site on the Sp110polypeptide that interacts with nuclear hormone receptors. A thirdgeneral approach is to interfere with binding of active Sp110 dimers toSp110-binding DNA sequences in the genome. One variation on thisapproach is to provide a molecule, e.g., a polypeptide, that binds to(blocks) Sp110-binding DNA sequences. Another variation on this approachis to provide an oligonucleotide based on an Sp110-binding DNA sequencein the genome. The oligonucleotide binds to (blocks) the DNA-bindingsite(s) on the Sp110 dimer.

Primary biliary cirrhosis (PBC) is an autoimmune disease thatpredominantly affects intrahepatic bile ducts. As with many otherautoimmune diseases, the vast majority of patients with PBC are womenand the etiology of this disorder is unknown. The natural history of PBCis one of slowly progressive cholestasis with the development ofcirrhosis and death unless the patient undergoes liver transplantation.Disease progression in an individual patient, however, is highlyvariable and a pre-symptomatic phase may last longer than 20 years(Springer et al., Am. J. Gastroenterol. 94:47-53, 1999 and Mahl et al.,J. Hepatol. 20:707-7 13, 1994). Because of the variable course ofpatients with PBC and the availability of novel treatments for thisdisease, it is important to identify prognostic factors that maydistinguish those patients with mild disease (who may not requiretreatment) from those with a more aggressive, rapidly progressive,illness.

The invention provides methods for identifying and characterizing novelautoantibody markers of disease state and prognosis in PBC patients.Serum samples from PBC patients are tested for the presence ofantibodies that react with Sp110. Various types of methods for detectingand quantifying such antibody-antigen reactions are known and can beemployed.

In some embodiments of the invention, PBC-related autoantibodies aredetected and characterized in methods such as conventionalimmunoblotting (Western) techniques, wherein an Sp110 polypeptide (orSp110 polypeptide fragment) is employed as an antigen. Typically, theSp110 polypeptide is subjected to SDS-polyacrylamide gel electrophoresis(SDS-PAGE) and blotted (transferred) onto a suitable membrane, e.g.,nitrocellulose, where the Sp110 antigen is immobilized. Sera from PBCpatients are diluted as necessary and contacted with the Sp110antigen-bearing membrane under suitable conditions for specific bindingof the immobilized antigen to an anti-Sp110 antigen, if one is present.After suitable washing steps, bound antibody is detected.

Detection of bound antibody can be accomplished by any suitable method.For example, labeled protein A, which binds to IgG with high specificityand high affinity, can be used. The protein A can be labeled in any ofvarious ways. Useful types of label include a conjugated calorimetricenzyme, e.g., horseradish peroxidase, a conjugated fluorochrome, e.g.,FITC, or a radioactive atom, e.g., ¹²⁵I. Immunoblot assay results can bequantitated, for example, by incorporating internal standards and asuitable optical scanning device. Preferably, suitable positive andnegative controls are employed in testing patient sera in addition toSp110, other nuclear body components such as Sp100, Sp140 polypeptidescan be tested simultaneously, e.g., on the same immunoblot membrane.

In some embodiments of the invention, PBC-related autoantibodies aredetected and quantitated using techniques designed for rapid,high-volume screening, e.g., microtiter plate ELISA techniques. Suchtechniques are known in the art and can be employed in practicing thepresent invention without undue experimentation.

EXAMPLES

The invention is further illustrated by the following experimentalexamples. The examples are provided for illustrative purposes only, andare not to be construed as limiting the scope or content of theinvention in any way.

Example 1 Isolation and Characterization of cDNA Clones Encoding Sp110

A nucleotide sequence in the EST database that encodes a polypeptidehomologous to the N-terminal portions of Sp110 and Sp140 was obtainedfrom the IMAGE consortium (accession number AA431918). This materialproved unsuitable to prepare probes for screening a cDNA library becauseit was highly contaminated with unrelated cDNAs. Accordingly, twooligonucleotides were synthesized based upon the sequence of the ESTclone (5′-TTGAATTCATGGAAGAGGCTCTTTTTCAG-3′ (SEQ ID NO:10) and5′-TTGAATTCCTTCTGCTAGGCCAGTTGG-3′ (SEQ ID NO:11)) and the polymerasechain reaction (PCR) was used to synthesize a fragment of the cDNA. ThePCR product was radiolabeled and used to screen a λGT10 cDNA libraryprepared from human spleen (Clontech, Palo Alto, Calif.). Six cDNAclones from among approximately one million bacteriophages hybridizedwith the radiolabeled probe and were isolated by plaque purification.Bacteriophage growth, DNA isolation, and subcloning into pUC19 wereperformed using standard procedures (Sambrook et al, 1989, MolecularCloning—A Laboratory Manual, 2^(nd) Ed., Cold Spring Harbor Press,1989). The nucleotide sequence of the full-length cDNA was determined bythe dideoxy chain termination method (Sanger et al., Science214:1205-1210, 1981). The sequences of the six clones, when assembled,revealed the sequence of the full length Sp110 cDNA (FIG. 1).

The cDNA encoding Sp110 was 2,337 base pairs long, with an open readingframe from nucleotides 78 to 2144 encoding a protein containing 689amino acids (FIGS. 1 and 3). The start codon was preceded by an in-framestop codon, indicating that this is a full-length cDNA. The amino acidresidues at 241 to 605 of Sp110 were essentially identical to residues 1to 365 of a previously reported polypeptide designated nuclearphosphoprotein 72 (Kadereit et al., J. Biol. Chem. 268: 24432-24441,1993). In this region, the amino acid sequence of Sp110 differed fromthat of nuclear phosphoprotein 72 at amino acid 580 (I-M).

The N-terminal portion of Sp110, between amino acid residues 6 and 159was 49% identical to the N-terminal portions of both Sp100 (Szostecki etal., J. Immunol. 145:4338-4347, 1990) and Sp140 (Bloch et al., J. Biol.Chem. 46:29198-29204, 1996). A second region of homology between Sp110and both Sp100b and Sp140 was present between amino acid residues 452and 532. In this region, Sp110 was 53% identical to Sp100b (Dent et al.,Blood 88:1423-1436, 1996) and 49% identical to Sp140. This portion ofSp100b and Sp140 was previously designated a SAND domain (Gibson et al.,Trends Biochem Sci. 23:242-244, 1998). Sp110 amino acid residues 537 to577 spanned a plant homeobox domain (Aasland et al., Trends Biochem.Sci. 20:56-59, 1995), and amino acid residues 606 to 674 contained theA, B, and C helices of a bromodomain (Jeanmougin et al., Trends BiochemSci. 22:151-153, 1997). The plant homeobox domain and bromodomain ofSp110 were 71% and 54% identical to the corresponding regions in Sp140.In addition, these portions of Sp110 were 56% and 46% identical to thecorresponding regions in murine TIF1α.

Example 2 Expression of Sp110 in Human Tissues and Cell Lines

The level of Sp110 mRNA in human tissues was determined by hybridizingmembranes containing 2.5 μg of poly(A)⁺-selected RNA from human tissues(multiple tissue Northern blots, Clontech Laboratories, Palo Alto,Calif.) with a ³²P-radiolabeled 1.4 kb XbaI restriction fragment of theSp110 cDNA. The human tissues represented on the membranes were spleen,thymus, prostate, testis, ovary, small intestine, colon, and peripheralblood leukocytes. The membranes were washed under stringent conditionsand exposed to autoradiographic film for one hour. To confirm thepresence of poly(A)⁺-selected RNA in each lane, the membranes werehybridized with a ³²P-radiolabeled β-actin cDNA probe. The membraneswere washed under stringent conditions and exposed to autoradiographyfilm for 30 minutes.

High levels of Sp110 mRNA were detected in human peripheral bloodleukocytes and spleen. In contrast, lower levels of Sp110 mRNA wereobserved in thymus, prostate, testis, ovary, small intestine, and colon.In addition, low levels of Sp110 mRNA were observed in human heart,brain, placenta, lung, liver, skeletal muscle, kidney, and pancreas.

To investigate the expression of Sp110 in cells of themonocyte/granulocyte lineage, RNA was prepared from the myeloidprecursor cell lines HL60 and NB4 (HL60 cells are available from theAmerican Type Culture Collection, Manassas, Va.). RNA was extracted fromthese cell lines using the guanidinium isothiocyanate-cesium chloridemethod (Sambrook et al., Cold Spring Harbor Laboratory, Cold SpringHarbor, NY, 1989). The HL60 cells were maintained in RPMI supplementedwith 10% fetal calf serum, L-glutamine (2 mM), penicillin (200units/ml), and streptomycin (200 mg/ml). RNA was fractionated informaldehyde-agarose gels (5 μg/lane) and equal loading of RNA wasconfirmed by staining 28S and 18S ribosomal RNA with ethidium bromide.RNA was transferred to nylon membranes and the membranes were hybridizedwith the radiolabeled XbaI restriction fragment of the Sp110 cDNA or theEcoRI/BamHI restriction fragment of the cDNA encoding human Sp100 (Blochet al., J. Biol. Chem. 46:29198-29204, 1996). Membranes were washed andanalyzed by autoradiography. Low levels of Sp110 mRNA were detected inboth NB4 cells and HL60 cells.

To examine the effect of cellular differentiation on Sp110 mRNA, NB4cells were treated for 48 hours with all trans retinoic acid (ATRA) (1μM. Following this treatment, the level of Sp110 mRNA was increased inNB4 cells, which indicates that differentiation of NB4 cells isassociated with increased expression of Sp110. To examine the effect ofIFN-γ treatment on Sp110 mRNA levels, HL60 cells were treated with IFN-γ(200 units/ml) for 48 hours. A marked increase in Sp110 mRNA wasobserved. These results demonstrated that, as with Sp100, PML, andSp140, IFN-γ-treatment enhances expression of Sp110.

Sp110 expression was also examined in human coronary artery smoothmuscle cells (hCASMCs) and human umbilical vein endothelial cells(HUVECs) following exposure to IFNα (200 u/ml). Expression of Sp110 mRNAwas induced within 4 hours and reached a maximum level between 8 and 24hours. Similar results were obtained after treating hCASMCs or HUVECswith IFNγ or IL-1β and TNFα. Sp110 gene expression was also markedlyinduced in hearts of mice treated with endotoxin, suggesting thatinflammatory mediators increase Sp110 mRNA levels in cardiac myocytes.

The observation that Sp110 was expressed in human leukocytes andcytokine-treated human vascular cells suggested that Sp110 is present incells that have important roles in the pathogenesis of atherosclerosis.

Example 3 Cellular Localization of Sp110

To study the cellular location of Sp110, antiserum directed against arecombinant fragment of Sp110 (amino acid residues 219 to 324) wasgenerated in rats, and an adenovirus vector encoding Sp110 (Ad.Sp110)was prepared.

To construct an E1-deleted, recombinant adenovirus vector containingSp110, the cDNA encoding Sp110 was cloned into the NotI and BamHI sitesof pAd.RSV₄, which contained the Rous sarcoma virus long-terminal repeatpromoter and the SV40 polyadenylation signal. The plasmid containingSp110 was co-transfected into 2093 cells with pJM17. Homologousrecombination between the two plasmids resulted in an adenovirus(Ad.Sp110) that contained Sp110 sequences in place of E1 sequences.Recombinant viruses in a plaque were amplified in 293 cells, and ahigh-titer stock was prepared. The 293 cells were grown in low glucose(1 g/L)-DMEM supplemented with 10% horse serum. The absence ofreplication-competent adenovirus in the viral stock was confirmed by thefailure of Ad.Sp110 to produce cytopathic changes in A549 lung carcinomacells. In addition, PCR failed to amplify a DNA fragment correspondingto the E1 region of adenovirus using oligonucleotides and the Ad.Sp110stock. An adenovirus-vector containing the cDNA encoding Sp140 wasdescribed previously in (Bloch, et al., Mol. Cell. Biol. 19:4423-4430,1999).

To produce antibodies directed against Sp110, three male Sprague-Dawleyrats were immunized with recombinant protein containing amino acidresidues 219435 of Sp110 fused to glutathione-S-transferase (GST). Theplasmid encoding this portion of Sp110 was prepared by ligating aBstYI/EcoRV restriction fragment of the cDNA encoding Sp110 into theBamHI/SmaI sites of pGEX (Pharmacia Biotech, Inc., Piscataway, N.J.).The plasmid was used to transform E. coli, and expression of the fusionprotein was induced by treatment withisopropyl-1-thio-β-D-galactopyranoside. The fusion protein was purifiedfrom E. coli proteins as described in (Smith et al., Gene 67:31-40,1988). Primary immunizations of three rats was performed using 50 μg ofpurified protein emulsified in complete Freund's adjuvant for eachanimal. Two subsequent booster injections consisting of 50 μg of proteinwere given at two-week intervals.

The rat anti-Sp110 antiserum reacted with Sp110 in extracts preparedfrom Ad.Sp110-infected HEp-2 cells, but not with Sp140 in extractsprepared from Ad.Sp140-infected HEp-2 cells or with Sp100, which isnormally expressed in HEp-2 cells. In contrast, rat anti-Sp140antiserum, previously prepared against amino acid residues 131′-391 ofSp140 (Bloch et al., J. Biol. Chem. 46:29198-29204, 1996), reacted withSp140, but not with Sp110 or Sp100. These results demonstrated that therat anti-Sp110 antiserum was specific for Sp110.

To investigate the cellular location of Sp110, rat anti-Sp110 antibodieswere used to stain NB4 cells before and after treatment with RA.Anti-Sp110 antiserum stained nuclear bodies in NB4 cells that weretreated for 48 hours with ATRA (1 μM), but did not react with untreatedNB4 cells.

To determine the location of Sp110 with respect to the PML/Sp100 nuclearbody, NB4 cells were treated with ATRA and stained with rat anti-Sp110antiserum and human serum containing antibodies directed against Sp100.Sp110 co-localized with Sp100 in nuclear bodies.

To further investigate the cellular location of Sp110,adenovirus-mediated gene transfer was used to express Sp110 in humancell lines in which it is not normally expressed. At a multiplicity ofinfection (MOI) of 25 viruses per cell, approximately 25% of HEp-2 cellsexpressed levels of Sp110 that were detectable by indirectimmunofluorescence. Surprisingly, Sp110 did not localize to nuclearbodies in these cells, but instead appeared to produce a granularnuclear staining pattern with prominent staining near the nuclearmembrane. Cytoplasmic staining was also observed in a few cells. Thecontrasting results obtained using the leukocyte cell line NB4 andAd.Sp110-infected HEp-2 cells can be reconciled with the present resultif Sp140, another leukocyte-specific nuclear body component, recruitsSp110 to the nuclear body. HEp-2 cells were infected with both Ad.Sp140,at an MOI of 50, and Ad.Sp110, at an MOI of 25. At an MOI of 50,essentially all of the HEp-2 cells expressed detectable Sp140 withinnuclear bodies. In cells infected with Ad.Sp140 alone, anti-Sp110antiserum did not stain nuclear bodies, confirming that anti-Sp110antiserum did not cross-react with Sp140. In cells infected with bothSp110 and Sp140, Sp110 localized to nuclear bodies and co-localized withSp100-containing nuclear bodies. These results demonstrated that Sp140enhances localization of Sp110 to the nuclear body.

Example 4 Transcriptional Activation by Sp110

The amino acid sequence motifs in Sp110, including, the SAND domain, thePHD, and the bromodomain, suggested that Sp110 has a role in theregulation of gene transcription. To examine the potential effect ofSp110 on gene transcription, a eukaryotic expression plasmid encodingSp110 fused to the DNA-binding domain of GAL4 (pBXG-Sp110) wasco-transfected with a CAT reporter plasmid containing five GAL4 bindingsites and an SV40 enhancer region (pG5SV-BCAT) into COS cells. There wasa dose-dependent increase in CAT activity in cells transfected withincreasing amounts of pBXG-Sp110. These results are similar to thoseobserved using pBXG-Sp140 and different from those obtained withpBXG-Sp100. The GAL4-Sp100 fusion protein was previously shown toinhibit CAT activity when co-transfected with the reporter plasmid(Seeler et al. Proc. Natl. Acad. Sci. USA 95:7316-7321, 1998; Lehming etal., Proc. Natl. Acad. Sci. USA 95:7322-7326, 1998; and Bloch, et al.,Mol. Cell. Biol. 19:4423-4430, 1999). These results demonstrated thatSp110 is capable of modulating gene transcription and acts in thesecells as a transcriptional activator.

Experiments were also performed to determine whether Sp110 couldfunction as a retinoic acid receptor (RAR) transcriptional co-activator.When co-transfected into COS cells with a reporter gene containing threecopies of the RARα response element, Sp110 significantly enhancedATRA-induced expression of the reporter gene. Similar results wereobserved in studies using HeLa cells instead of COS cells. The extent ofreporter gene activation by Sp110 was similar to that induced by thenuclear body component PML. These results demonstrated that Sp110 canfunction as a co-activator of the nuclear hormone receptor RAR

Sp110 also acted as a co-activator of PPARα. When COS cells wereco-transfected with PPARα and a reporter gene containing three copies ofthe PPAR response element (PPRE), Sp110 markedly enhancedagonist-induced expression of the reporter gene compared with the effectof PPARα expressed alone. Sp110 may interact with nuclear hormonereceptors via an LXXLL domain. To determine whether Sp110 interacts withPPARα via the LXXLL domain, oligonucleotides and PCR were used toprepare a mutant Sp110 protein (Sp110m) in which two of the threeleucine residues were changed, one to valine and one to alanine (LXXVA).

Although immunoblot studies demonstrated that Sp110 and Sp110m wereexpressed at similar levels in transfected COS cells, the Sp110 mutantactivated the PPARα receptor less than wild-type Sp110. These resultsdemonstrated that Sp110 functions as a PPAR transcriptional co-activatorand indicated that the interaction between Sp110 and PPARα may involvethe LXXLL nuclear hormone receptor interaction domain.

Sp110 enhanced signal transduction through the retinoic acid receptor α(RARα) (LaMorte et al., Proc Natl Acad Sci USA 95:4991-4996, 1998) and,in contrast to the results obtained with PPARα, expression of Sp110mwith RARα also enhanced expression of the reporter gene. Similarly, bothSp110 and the Sp110 mutant enhanced signal transduction through thePPARγ receptor. These results suggested that Sp110 had an effect on RARαsignaling and PPARγ signaling, perhaps through interaction withnucleotide motifs adjacent to nuclear hormone receptor responseelement(s). Although the examples below involved the PPARα receptor, itis predicted that similar results will be achieved with other nuclearhormone receptors.

Example 5 PPARα Interaction with CBP/p300

PPARα interacted with the N-terminal portion of CBP/p300 between aminoacid residues 39 and 221. To determine whether Sp110 also interacts withCBP, the mammalian two-hybrid system was used as an assay. In thisassay, the GAL4 DNA-binding domain (GAL4) fused to CBP (GAL4CBP) and aluciferase reporter gene containing GAL4 response elements wereexpressed in COS cells with either Sp110 fused to the herpes simplexvirus VP16 activation domain (VP16-Sp110) or VP16 alone. Expression ofVP16-Sp110 significantly enhanced Gal 4CBP-induced luciferase geneactivity compared with VP16 alone. Thus, Sp110 interacts, eitherdirectly or indirectly, with CBP. The site of functional interactionbetween Sp110 and CBP was mapped more specifically to amino acidresidues 271-720 of CBP. The Sp110-CBP functional interaction domain istherefore distinct from the PPARα-CBP interaction domain.

To identify the portion of Sp110 that functionally interacts with CBP,the N-terminal or C-terminal portions of Sp110 were fused to VP16 andexpressed in COS cells together with GAL4CBP and a reporter gene. TheC-terminal portion, which contained the PHD and the bromodomain,enhanced GAL4CBP-induced expression of the reporter gene, but theN-terminal portion, which contained the Sp100-like region, did not. Asdescribed below, studies can be carried out to further delineate thedomains that mediate functional interaction between Sp110 and CBP and totest whether Sp110 interacts with CBP directly.

Sp110 does not contain motifs, such as acetyltransferase (as inCBP/p300) or kinase domains (as in TIF1α), that are known to activategene transcription. To identify the portion of Sp110 that enhancesreporter gene expression, DNA segments encoding fragments of Sp110 fusedto GAL4 were expressed with a reporter gene in COS cells. Neither theN-terminal (Sp100-like domain), nor the C-terminal (PHD/bromodomain)portions of Sp110 increased expression of the reporter gene. Incontrast, fusion proteins containing the middle portion of the protein(putative activation domain and SAND domain) increased reporter geneexpression. The mechanism by which Sp110 enhances PPARα-mediated genetranscription can be investigated, as described below, by identifyingproteins that interact with the activation domain and SAND domain.

Example 6 Sp110 and Expression of Genes Regulated by PPARα

This experiment tests the possibility that Sp110 enhances expression ofa reporter gene under the control of the native promoter region of CPTI,a gene that is regulated by PPARα. Co-transfection studies are performedin COS cells using a luciferase reporter construct that contains a CPTIgene promoter having the PPRE (AGGGAAaAGGTCA; SEQ ID NO:). The effect ofco-expression of Sp110 and PPARα on luciferase activity is compared tothat of Sp110, PPARα, or control vectors alone. An expected result isthat Sp110 enhances PPARα-mediated expression of the reporter gene underthe control of the CPT I promoter region.

Sp110 may enhance luciferase expression by interacting with otherregulatory elements within the CPTI promoter region. To demonstrate thatthe effect of Sp110 on luciferase activity requires PPARα, the effect ofSp110 on a luciferase reporter plasmid that has a mutated PPRE(AGGGAAaAccTCA; SEQ ID NO:), is determined. If Sp110 enhances CPTIpromoter-driven luciferase activity by interacting with PPARα, then anexpected result is that Sp110 has no effect on expression of thereporter plasmid with the mutated PPRE.

Example 7 Sp110 and PPARα Cooperation in Mitochondrial Fatty AcidOxidation Gene Expression

The fibroblast cell line 3T3-L1 can be induced to differentiate intocells that resemble white adipocytes. These cells normally express lowlevels of enzymes involved in mitochondrial fatty acid oxidation. Totest the possibility that Sp110 enhances PPARα-mediated upregulation ofmitochondrial fatty acid oxidation gene expression, Sp110 isoverexpressed in 3T3-L1 cells using an adenovirus vector (Ad.Sp110). Anunrelated protein, green fluorescent protein (GFP), is expressed as acontrol using a second adenovirus vector (Ad.GFP). 3T3-L1 cells will beinfected with Ad.Sp110 or Ad.GFP at a virus MOI sufficient to produceinfection of more than 90% of the cells. The cells are then incubated inmedium containing insulin, 3-isobutyl-1-methylxanthine (IBMX), anddexamethasone to induce differentiation (Cao et al., Genes Dev.,5:1538-1552, 1991). Immunobloting is performed to confirm successfultransgene expression. RNA blot hybridization will be performed tomeasure the effect of Sp110 (compared with GFP) on expression of threefatty acid oxidation genes (MCAD, LCAD, and CPT-I) in the presence orabsence of the PPARα agonist WY 14,643.

To examine the effect of Sp110 on the rate of fatty acid oxidation, 3T3L1 cells are infected with either Ad-Sp110 or Ad.GFP, and the rate ofpalmitate oxidation is determined (Gulick et al., Proc. Natl. Acad.Sci., USA, 91:11012-11016, 1994). Seventy-two hours after infection, thecells are incubated with [¹⁴C] palmitate. The tissue culture platescontain a central well with a piece of filter paper. After 6 hours, the¹⁴CO₂ is released from tissue culture medium by addition of 6 N HCl and¹⁴CO₂ is collected overnight by alkalinization of the filter paper with2 N NaOH. ¹⁴CO₂ is measured by scintillation counting of the filters.

Mutations in the LXXLL domain of Sp110 impair its ability to enhancePPARα-mediated expression of a reporter gene, most probably by blockingthe direct interaction between PPARα and Sp110. To investigate theimportance of the LXXLL domain in enhancing PPARα mediated geneexpression, an adenovirus vector encoding Sp110m (Ad.Sp110m) is testedfor its ability to induce expression of MCAD, LCAD, and CPT-I and toincrease fatty acid oxidation in 3T3-L1 cells. The results of thesetests with Ad.Sp110m are directly compared with those obtained usingAd.Sp110.

An expected result is that Sp110, but not GFP, enhances Wy14,643-induced (PPARα agonist-induced) expression of genes involved infatty acid oxidation and increase palmitate oxidation in 3T3-L1 cells.Because the effect of Sp110 on PPARα is expected to require directinteraction between the two proteins, Sp110 is expected to be moreeffective than Sp110m at producing these changes. If Sp110 and Sp110mare equally effective at inducing fatty acid oxidation, the putativeLXXLL interaction domain in Sp110 is deemed non-crucial for this effect.In that event, studies to define alternative interaction sites betweenPPARα and Sp110 could be carried out.

3T3-L1 cells express relatively low levels of PPARα. In fact, theselevels may be too low to detect any effect of Sp110 on genes normallyregulated by PPARα. Thus, if there appears to be no enhancement ofexpression of genes involved in fatty acid oxidation following infectionwith Ad.Sp110, the studies are performed using an adenovirus vectorencoding PPARα. Successful production of PPARα is confirmed usingimmunoblots and a commercially available anti-PPARα antibody. 3T3-L1cells will be infected with Ad.GFP, Ad.Sp110, Ad.PPARα, or both Ad.Sp110and Ad.PPARα. The effect of Ad.PPARα and Ad.Sp110 (together) on theexpression of fatty acid oxidation genes and palmitate oxidation ratesis compared to the effect of Ad.Sp110, Ad.PPARα, and Ad.GFP alone.

Example 8 Expression of Sp110 in hCASMCs and IL-1-Induced Production ofIL-6 and Cyclooxygenase (COX)-2

Inflammatory cytokines such as IL-1 induce expression of IL-6 and COX-2in smooth muscle cells (SMCs). This expression can be blocked, however,if the SMCs are treated with PPARα agonists (Staels et al., J. Clin.Invest. 103:1489-1498, 1999). Endogenous Sp110 expression in SMCs wasenhanced by treatment with inflammatory cytokines. Thus, Sp110 isexpected to enhance PPARα-mediated inhibition of the inflammatoryresponse in SMCs.

Forty-eight hours after human coronary artery SMCs (hCASMCs) areinfected with Ad.Sp110, they are treated with recombinant human IL-1 andWy14,643. The amount of IL-6 released by SMCs is measured byradioimmunoassay. To confirm that the effect of Ad.Sp110 onPPARα-mediated inhibition of IL-6 production is not a result of the Advector alone, control cells are infected, in parallel, with Ad.GFP atthe same MOI as used for Ad.Sp110. Cells are treated with IL-1 andWy14,643 and IL-6 production are measured as described above.

Because Sp110 also is expected to augment the ability of PPARα toinhibit COX-2 gene expression, SMCs are infected with Ad.Sp110 andsubsequently treated with IL-1 as described above. The cells are treatedwith increasing amounts of the PPARα agonist Wy14,643 and theconcentration of COX-1 and COX-2 protein will be measured usingimmunoblot techniques. Parallel experiments are conducted with Ad.GFP asa control.

While the concentration of COX-1 is expected to be unaffected either byexpression of Sp110 or by treatment with IL-1 or WY14,643, theconcentration of COX-2 is expected to change. A lower concentration ofCOX-2 at each dose of WY14,643 in Ad.Sp110-infected cells (relative toAd.GFP-infected cells), would suggest that Sp110 augments the ability ofPPARα to inhibit COX-2 expression in SMCs in response to IL-1.

Example 9 Inhibiting PPARα-Sp110 Interaction in Cytokine-Treated hCASMCs

The biological function of Sp110 can be assessed in numerous ways,including studies in which it is overexpressed (as can be done toinvestigate its role in PPARα-mediated signal transduction) and studiesin which oligopeptides are used as inhibitors (as can be done to inhibitthe ligand-dependent interaction between PPARα and Sp110).

Two complementary oligonucleotides encoding a peptide that spans sevenamino acids on either side of the LXXLL domain of Sp110 are synthesizedand ligated, in frame, to GAL4 in a eukaryotic expression plasmid(pGAL-LXXLL). To demonstrate that the GAL4-oligopeptide fusion proteinacts an inhibitor, this expression plasmid are co-transfected into COScells with plasmids encoding PPARα, Sp110, and a reporter gene constructin which the reporter is driven by PPRE. As a control, cells aretransfected in parallel with a plasmid encoding pGAL alone. The use ofGAL4 as a fusion partner facilitates nuclear localization of theoligopeptide. In addition, successful production of theGAL4-oligopeptide fusion protein in transfection assays is confirmed byimmunoblotting with anti-GAL4 antibodies. Expression of theGAL4-oligopeptide in COS cells is expected to block agonist-specific,Sp110-mediated, activation of PPARα.

To examine the effect of inhibiting the interaction between PPARα andSp110 in hCASMCs, an adenovirus vector encoding GAL4-LXXLL and a controlvirus encoding GAL4 fused to the same amino acid residues in randomrearrangement are prepared. hCASMCs are treated with IL-1 andsubsequently infected with either Ad.GAL4-LXXLL or the controladenovirus vector. The production of IL-6 and the induction of COX-2 areassayed as described above.

Treatment of hCASMCs with cytokines induces expression of Sp110.Overexpression of an oligopeptide corresponding to the LXXLL domain inSp110 would be expected to block Sp110 coactivation of PPARα-mediatedgene expression. Thus, the oligopeptide is expected to enhance thecytokine-mediated induction of IL-6 and COX-2.

Example 10 Sp110 Interaction with PPARα

Mutations in the putative nuclear hormone receptor interaction domain ofSp110 inhibit the ability of Sp110 to enhance PPARα-mediatedtranscriptional activity. These results suggest that the LXXLL domain inSp110 interacts with PPARα. It is possible, however, that other portionsof Sp110 also mediate its interaction with PPARα. Mammalian two-hybridassays are used to identify the portions of Sp110 and PPARα that mediatefunctional interaction between these two proteins.

To identify the portion(s) of Sp110 that mediate interaction with PPARα,DNA molecules encoding portions of Sp110 (the Sp100-like region,putative activation domain, SAND domain, LXXLL domain, PHD, andbromodomain, individually and in combination) are ligated into theeukaryotic expression vector pVP16 in-frame with the HSV VP16 activationdomain. Each of the resulting plasmids are co-transfected into COS cellswith a second plasmid encoding PPARα and a reporter gene containing aPPRE. Successful production of each fragment of Sp110 is confirmed usingimmunoblots and an antibody directed against VP16. The ability of eachVP16-Sp110 fusion protein to enhance reporter gene expression in thepresence of PPARα and agonist provides evidence for a functionalinteraction between a given Sp110 portion and PPARα. Control experimentsare conducted to rule out the possibility that VP16-Sp110 fusionproteins activate the reporter gene in the absence of PPARα. Inaddition, the inability of PPARα and VP16-Sp110 fusion proteins toactivate reporter gene expression from a plasmid that lacks the PPREwill be confirmed.

To identify portions of PPARα that mediate interaction with Sp110, DNAmolecules encoding portions of PPARα will be ligated into a eukaryoticexpression plasmid in-frame with GAL4. The plasmids will beco-transfected with a plasmid encoding-Sp110 fused to VP16 and areporter gene with an upstream GAL4 response element. Gene expressionmediated by GAL4-PPARα-fragment and Sp110 will be compared to thatmediated by the same GAL4-PPARα-fragment and control plasmid. Theability of VP16-Sp110 to enhance GAL4-PPARα-fragment-induced reportergene expression will be evidence of an interaction between Sp110 and aPPARα fragment.

These studies will identify the portions of Sp110 and PPARα that mediatefunctional interaction between the two proteins. Among others, theinteraction domains should include, the LXXLL domain of Sp110 and theligand-binding “activation function 2” (AF2) portion of PPARα.

Example 11 Sp110 Direct Interaction with PPARα

Sp110 is predicted to interact directly with PPARα. A GST-Sp110 fusionprotein is prepared and tested in vitro for interaction with³⁵S-radiolabeled PPARα DNA encoding Sp110 is ligated in-frame with DNAencoding glutathione-S-transferase (GST) in the prokaryotic expressionplasmid pGEX so as to encode a GST-Sp110 fusion protein. The recombinantfusion protein is expressed in E. coli, affinity-purified, andimmobilized on Sepharose beads. DNA encoding PPARα are used to preparedialed protein by in vitro transcription and translation and will beincubated with Sepharose-GST-Sp110 (or Sepharose-GST alone) in thepresence and absence of PPARα agonist. Bound and radiolabeled PPARα iseluted from Sepharose-GST-Sp110 (or Sepharose-GST) by boiling inSDS/PAGE sample buffer, and the eluant will be fractionated by SDS/PAGE.

Retention of PPARα on Sepharose-GST-Sp110, but not on controlSepharose-GST, provides evidence for a direct interaction between PPARαand Sp110. In addition, if PPARα is retained on Sepharose-GST-Sp110 inthe presence, but not in the absence, of PPARα agonist, then the directinteraction between PPARα and Sp110 requires the presence of nuclearhormone receptor agonist.

Example 12 Sp110 Interaction with CBP

A mammalian two-hybrid assay was used to demonstrate a functionalinteraction between the PHD/bromodomain of Sp110 and amino acids 271-720of CBP. Sp110 may also interact directly with CBP. To determine whetheror not it does, a GST-CBP (271-720) fusion protein will be prepared (asdescribed above for Sp110) and exposed to ³⁵S-radiolabeled Sp110. An³⁵S-radiolabeled nuclear body component, PML, can serve as a positivecontrol for interaction with CBP, as PML interacts with CBP in thisportion of the protein (Doucas et al., Proc. Natl. Acad. Sci. USA,96:2627-2632, 1999). If CBP interacts directly with Sp110, then aSepharose-GST-CBP (271-720) fusion protein, but not Sepharose-GST alone,would be expected to retain radiolabeled Sp110.

CBP has been described as a “platform” protein because it interacts withmany other proteins. Among the proteins that interact with CBP at aminoacid residues 271-720 are: RXR, STAT2, CREB, JUN, MYB, ELK1, SREBP, andSAP1A (Giles et al., Trends Genetics, 14:178-183, 1998). Instead of adirect interaction between Sp110 and CBP, at least one other protein maymediate the functional interaction between these two proteins. If adirect interaction between Sp110 and CBP cannot be demonstrated usingGST pulldown experiments, then the mammalian two-hybrid system will beused to further delineate the site in CBP that mediates functionalinteraction with Sp110. Two approaches can then be taken to identify theprotein(s) that link CBP and Sp110. A candidate gene approach willinvolve obtaining cDNAs encoding proteins that are known to interactwith the identified portion of CBP. Proteins encoded by these cDNAs willbe tested for interaction with Sp110 using a combination of GSTpull-down and mammalian two-hybrid assays. If none of the candidategenes interacts with Sp110, then the yeast two-hybrid system will beused to identify cDNAs encoding proteins that link CBP and Sp110. A DNAfragment encoding the PHD/bromodomain of Sp110, and a DNA fragmentencoding the portion of CBP that mediates functional interaction withSp110, will each be used to screen a human leukocyte cDNA library.Complementary DNAs encoding interacting proteins will be divided intogroups that interact with Sp110, CBP, or both. Verification of “true”protein-protein interactions are then performed, as described below.

Example 13 Sp110 SAND Domain and Transcriptional Activation

Sp110 appears to enhance expression of nuclear hormone responsive genesby binding to a nucleotide sequence adjacent to the nuclear hormoneresponse element and directly or indirectly enhancing gene expression.The sand domain of Sp110 may mediate DNA binding. By interactingdirectly with DNA, Sp110 may enhance gene expression independent of theLXXLL motif. A GAL4-Sp110 fusion protein is capable of activatingexpression of a reporter gene driven by the GAL4 response element, andneither the is N-terminal, which contains the Sp100-like domain, nor theC-terminal, which contains the LXXLL/PHD/bromodomain) of Sp110 arerequired to mediate enhanced expression of the reporter gene. The middleportion of Sp110 contains a SAND domain, an amino acid sequence motifthat has been observed in several proteins that regulate genetranscription.

To identify and characterize cDNAs encoding proteins that interact withSp110's activation domain, a DNA fragment encoding that domain will beused as “bait” in the yeast two-hybrid assay. The DNA fragment areligated into plasmid pGBKT7 (Clontech) and transferred into yeast with aleukocyte cDNA library prepared in pGADT7. The transformed yeast will bescreened for the ability to grow on His⁻ medium and colonies that growon this medium will be tested for α-galactosidase (α-gal) activity.Complementary DNAs are recovered from positive yeast clones andre-tested for interaction with bait by a second round of transformationin yeast. If numerous positive clones are obtained, they will be sortedinto groups on the basis of size and restriction sites, andrepresentative clones will be tested for interaction with an unrelatedbait fusion protein. Clones encoding proteins that specifically interactwith the Sp110 activation domain, but not the unrelated protein, will besequenced and further characterized

In vitro co-immunoprecipitation, in vivo co-immunoprecipitation andMammalian two-hybrid assays will be used to confirm functionalinteractions between Sp110 and proteins produced by cDNAs identifiedusing yeast two-hybrid. These studies identify cDNAs encoding proteinsthat interact with the activation domain of Sp110. The identity of theseproteins will provide additional information regarding the mechanism bywhich Sp110 functions as a nuclear hormone receptor co-activator.

In an alternative approach, proteins that interact with the activationdomain are identified by purifying interacting proteins from cellextracts using recombinant Sp110 protein linked to Sepharose beads.Purified proteins will be fractionated in polyacrylamide gels,transferred to IMMOBILON™ filters, and subjected to amino acidmicrosequencing. These techniques have been used previously to purifyand identify the predominant autoantigen in patients with autoimmunesensorineural hearing loss (Bloch et al., Archives ofOtolaryngology—Head and Neck Surgery 121:1167-1171, 1995).

Example 14 Sp110 in PBC Diagnosis

This study demonstrates that antibodies directed against nuclear bodycomponents Sp140 and Sp110 identify a subset of PBC patients with avariant form, e.g., a relatively mild form, of PBC. Serum from awell-defined cohort of 370 PBC patients are tested by immunoblot forantibodies directed against Sp110 and Sp140.

Adenovirus vectors containing cDNAs encoding Sp140, Sp110, and Sp100produce high levels of protein in human 293 cells. Aliquots of celllysates sufficient to screen 5,000 sera for each of these antigens areprepared. Cell lysates are boiled in loading buffer, fractionated in an8% polyacrylamide gel, and transferred to nitrocellulose membranes. Aminiblot apparatus is applied to the membrane. This permits screening of20 sera on each membrane. As a preliminary screen, patient serum isdiluted 1:200 in PBS containing 5% nonfat dry milk and incubated for onehour at room temperature with the nitrocellulose. Membranes are washedthree times with PBS and incubated with horseradish peroxidase (HRP)conjugated-protein A diluted 1:5000 in PBS. Filters are washed threetimes in PBS, incubated with chemiluminescence reagent, and then exposedto film. If a serum sample appears to contain antibodies directedagainst the recombinant protein, then a second immunoblot is performed.This second membrane has two lanes, one containing protein from 293cells infected with adenovirus encoding the nuclear body protein, andthe second containing protein from cells infected with the controladenovirus. The presence of an appropriate size band in the former lane,but not the latter, is taken as confirmation of the presence ofautoantibodies directed against the nuclear body component. This secondimmunoblot excludes false-positive results that may be secondary tohuman antibodies reacting with 293 cell proteins or adenovirus proteinsproduced in these cells.

The diagnosis of PBC in the 370-patient cohort is established based onthe presence of abnormal liver function tests, AMA (titer≧1:20) andliver biopsy compatible with PBC. The findings on liver biopsy areclassified into four stages (portal hepatitis, periportal hepatitis,septal fibrosis and/or bridging necrosis, cirrhosis). The cohortincludes 234 women and 36 men. The date of presentation was defined asthe first documentation of abnormal liver enzymes. The median age atpresentation is 52 years (range 24-81). The median length of follow-upis nine years (range 1-27). Symptoms of PBC in these patients includepruritis, jaundice, portosystemic encephalopathy, bleeding varices,edema and ascites. Endpoints of the study are liver transplantation,death from liver disease and death from other causes.

To demonstrate that antibodies directed against Sp110 and Sp140 identifya subset of patients with a mild form of PBC, serum from these patientsare screened for the presence of antibodies using immunoblot asdescribed above. For the purpose of this study, mild disease is definedas liver biopsy at presentation showing stage I or II disease andsurvival during the course of the study without requiring livertransplantation.

Other Embodiments

A number of embodiments of the invention have been described.Nevertheless, it is understood that various modifications may be madewithout departing from the spirit and scope of the invention. Otherembodiments are within the scope of the claims that follow.

1. An isolated DNA comprising a nucleotide sequence whose complementhybridizes under stringent hybridization conditions to a DNA moleculewhose nucleotide sequence consists of nucleotides 405 to 797 of SEQ IDNO:1.
 2. The DNA of claim 1, wherein the isolated DNA further comprisesa nucleotide sequence encoding a domain selected from the groupconsisting of: a domain having at least 80% sequence identity with aminoacids 6-109 of SEQ ID NO:2 (Sp100-like domain); a domain having at least80% sequence identity with amino acids 454-532 of SEQ ID NO:2 (SANDdomain); a domain having at least 80% sequence identity with amino acids537-577 of SEQ ID NO:2 (plant homeobox domain); and a domain having atleast 80% sequence identity with amino acids 606-674 of SEQ ID NO:2(bromodomain).
 3. The DNA of claim 1, wherein the isolated DNA furthercomprises a nucleotide sequence encoding a domain selected from thegroup consisting of: amino acids 6-109 of SEQ ID NO:2 (Sp100-likedomain) or amino acids 6-109 of SEQ ID NO:2 with one or moreconservative amino acid substitutions therein; amino acids 454-532 ofSEQ ID NO:2 (SAND domain) or amino acids 454-532 of SEQ ID NO:2 with oneor more conservative amino acid substitutions therein; amino acids537-577 of SEQ ID NO:2 (plant homeobox domain) or amino acids 537-577 ofSEQ ID NO:2 with one or more conservative amino acid substitutionstherein; and amino acids 606-674 of SEQ ID NO:2 (bromodomain) or aminoacids 606-674 of SEQ ID NO:2 with one or more conservative amino acidsubstitutions therein.
 4. The DNA of claim 1, wherein the isolated DNAfurther comprises a nucleotide sequence encoding: amino acids 6-109 ofSEQ ID NO:2 (Sp100-like domain) or amino acids 6-109 of SEQ ID NO:2 withone or more conservative amino acid substitutions therein; amino acids454-532 of SEQ ID NO:2 (SAND domain) or amino acids 454-532 of SEQ IDNO:2 with one or more conservative amino acid substitutions therein;amino acids 537-577 of SEQ ID NO:2 (plant homeobox domain) or aminoacids 537-577 of SEQ ID NO:2 with one or more conservative amino acidsubstitutions therein; and amino acids 606-674 of SEQ ID NO:2(bromodomain) or amino acids 606-674 of SEQ ID NO:2 with one or moreconservative amino acid substitutions therein.
 5. An isolated DNAcomprising a nucleotide sequence that encodes a polypeptide whose aminoacid sequence the sequence set forth as SEQ ID NO:2 or the sequence setforth as SEQ ID NO:2, with one or more conservative amino acidsubstitutions therein.
 6. The DNA of claim 1, wherein the isolated DNAcomprises a nucleotide sequence encoding a polypeptide comprising: aminoacids 6-109 of SEQ ID NO:2 (Sp100-like domain) or amino acids 6-109 ofSEQ ID NO:2 with one or more conservative amino acid substitutionstherein; amino acids 537-577 of SEQ ID NO:2 (plant homeobox domain) oramino acids 537-577 of SEQ ID NO:2 with one or more conservative aminoacid substitutions therein; and amino acids 606-674 of SEQ ID NO:2(bromodomain) or amino acids 606-674 of SEQ ID NO:2 with one or moreconservative amino acid substitutions therein; wherein the polypeptidedoes not contain a SAND domain.
 7. An isolated DNA comprising anucleotide sequence that encodes the amino acid sequence set forth asSEQ ID NO:5, or the sequence set forth as SEQ ID NO:5 with one or moreconservative amino acid substitutions therein.
 8. A vector comprisingthe DNA of claim
 1. 9. The vector of claim 8, wherein the DNA isoperably linked to an expression control sequence.
 10. A cell comprisingthe vector of claim
 8. 11-14. (canceled)
 15. An antibody that bindsspecifically to an Sp110 polypeptide.
 16. The antibody of claim 15,further comprising a detectable label.
 17. A screening method foridentifying a compound that inhibits Sp110 dimerization, the methodcomprising: providing an Sp110 polypeptide sample solution; adding tothe sample solution a candidate compound; and detecting a decrease inSp110 dimerization in the presence of the candidate compound, ascompared to Sp110 dimerization in the absence of the candidate compound,as an indication that the candidate compound inhibits Sp110dimerization.
 18. A screening method for identifying a compound thatenhances Sp110 dimerization, the method comprising: providing an Sp110polypeptide sample solution; adding to the sample solution a candidatecompound; and detecting an increase in Sp110 dimerization in thepresence of the candidate compound, as compared to Sp110 dimerization inthe absence of the candidate compound, as an indication that thecandidate compound enhances Sp110 dimerization.
 19. A screening methodfor identifying a polypeptide that dimerizes with Sp110 to form aninactive heterodimer, the method comprising: providing an Sp110polypeptide sample solution; adding to the sample solution a candidatepolypeptide, thereby forming a test mixture; providing a gene expressionsystem comprising a reporter gene operably linked to an Sp110-responsiveexpression control sequence; contacting the test mixture with the geneexpression system; detecting a decrease in reporter gene expressionlevel in the presence of the test mixture, as compared to geneexpression level in the presence of the Sp110 polypeptide samplesolution, as an indication that the candidate compound dimerizes withSp110 to form an inactive heterodimer.
 20. A screening method foridentifying a polypeptide that dimerizes with Sp110 to form aconstitutively active or hyperactive heterodimer, the method comprising:providing an Sp110 polypeptide sample solution; adding to the samplesolution a candidate polypeptide, thereby forming a test mixture;providing a gene expression system comprising a reporter gene operablylinked to an Sp110-responsive expression control sequence; contactingthe test mixture with the gene expression system; detecting constitutiveexpression or an increase in reporter gene expression level in thepresence of the test mixture, as compared to gene expression level inthe presence of the Sp110 polypeptide sample solution, as an indicationthat the candidate compound dimerizes with Sp110 to form aconstitutively active or hyperactive heterodimer.
 21. A screening methodfor identifying a compound or polypeptide that inhibits Sp110 binding toa nuclear hormone receptor, the method comprising: providing an Sp110polypeptide sample solution; adding to the sample solution a candidatecompound; adding to the sample solution a nuclear hormone receptor; anddetecting a decrease in Sp110 binding to the nuclear hormone receptor inthe presence of the candidate compound, as compared to Sp110 binding tothe nuclear hormone receptor in the absence of the candidate compound.22. A screening method for identifying a compound or polypeptide thatenhances Sp110 binding to a nuclear hormone receptor, the methodcomprising: providing an Sp110 polypeptide sample solution; adding tothe sample solution a candidate compound; adding to the sample solutiona nuclear hormone receptor; and detecting an increase in Sp110 bindingto the nuclear hormone receptor in the presence of the candidatecompound, as compared to Sp110 binding to the nuclear hormone receptorin the absence of the candidate compound.
 23. A screening method foridentifying a compound or polypeptide that inhibits the binding of anSp110 dimer to an Sp110-binding nucleotide sequence, the methodcomprising: providing an Sp110 polypeptide sample solution; adding tothe sample solution a candidate compound or polypeptide; adding to thesample solution an Sp110-binding nucleotide sequence; and detecting adecrease in Sp110 binding to the Sp110-binding nucleotide sequence inthe presence of the candidate compound, as compared to Sp110 binding tothe Sp110-binding nucleotide sequence in the absence of the candidatecompound.
 24. A screening method for identifying a compound orpolypeptide that enhances the binding of an Sp110 dimer to anSp110-binding nucleotide sequence, the method comprising: providing anSp110 polypeptide sample solution; adding to the sample solution acandidate compound or polypeptide; adding to the sample solution anSp110-binding nucleotide sequence; and detecting an increase in Sp110binding to the Sp110-binding nucleotide sequence in the presence of thecandidate compound, as compared to Sp110 binding to the Sp110-bindingnucleotide sequence in the absence of the candidate compound.
 25. Amethod for diagnosing primary biliary cirrhosis (PBC) in a humanpatient, the method comprising: providing a substantially pure Sp110polypeptide; providing a serum sample from the patient; contacting Sp110polypeptide with the serum sample; and detecting specific binding of anantibody in the serum with the Sp110 polypeptide as an indication ofPBC.
 26. A method for assessing the prognosis of a patient diagnosed ashaving primary biliary cirrhosis (PBC) in a human patient, the methodcomprising: providing a substantially pure Sp110 polypeptide; providinga serum sample from the patient; contacting Sp110 polypeptide with theserum sample; and detecting specific binding of an antibody in the serumwith the Sp110 polypeptide as an indication of the patient's prognosis.